Pseudomonas aeruginosa is an opportunistic pathogen affecting humans and is responsible for up to 20% of nosocomial infections and 3-6% of community-acquired infections. See Bodey, et al., Rev. Infect. Dis. 5(2):279-313 (1983). Individuals afflicted by certain medical conditions such as cystic fibrosis, leukemia, neutropenia, and burn wounds appear to be predisposed to Pseudomonas aeruginosa infections. See Bodey, et al., Rev. Infect. Dis. 5(2):279-313 (1983); Wood, Hosp. Pract. 11:91-100 (1976).
Pseudomonas aeruginosa produces both cell-associated and secreted factors which contribute to its pathogenesis. Nicas, et al., Can. J. Microbiol. 31:387-392 (1985). One class of virulence factors produced by Pseudomonas aeruginosa is the ADP-ribosyltransferases. Pseudomonas aeruginosa produces two ADP-ribosyltransferases, exotoxin A and exoenzyme S, which can be differentiated biochemically by target protein specificity, Coburn, et. al., Infect. Immun. 57(3):996-8 (1989), Coburn, et al., Infect. Immun. 59(11):4259-62 (1991), Iglewski, et al., Proc. Natl. Acad. Sci. U.S.A. 72(6):2284-8 (1975), Iglewski, et al., Proc. Natl. Acad. Sci. U.S.A. 75(7):3211-5 (1978); by the amino acid residue of the target protein that is ADP-ribosylated, Coburn, et al., J. Biol. Chem. 264(15):9004-8 (1989); Iglewski,et al., ADP-ribosylation of elongation factor 2 in animal cells. p. 511-524 (1990) In Moss and Vaughan (ed.), ADP-Ribosylating Toxins and G Proteins: Insights into Signal Transduction. American Society for Microbiology, Washington D.C.; and by their requirements for activation of enzymatic activity in vitro, Iglewski, et al., Proc. Natl. Acad. Sci. U.S.A. 75(7):3211-5 (1978) and Coburn, et al., J. Biol. Chem. 266(10):6438-46 (1991).
Exoenzyme S has been implicated as a mediator of Pseudomonas aeruginosa pathogenesis in burn wounds and chronic lung infections. Tn-1 mutagenesis of Pseudomonas aeruginosa strain 388 produced a mutant strain termed 388 exs1::Tn1 which lacked detectable exoenzyme S activity in culture supernatant fluids. See Nicas, et al., Infect. Immun. 45(2):470-4 (1984). In the burned mouse model, strain 388 exs1::Tn1 was greater than 2,000-fold less toxic than the wild type strain 388. Strain 388 exs1::Tn1 was not defective in its ability to colonize burn wounds, but showed decreased ability to disseminate from burn wounds when compared to the wild type strain 388. Antibody prepared against exoenzyme S protected burned mice against challenge with wild type strain 388. Nicas, et al., Antibiot. Chemother. 36(40):40-8 (1985).
In a chronic rat lung infection model, strain 388 exs1::Tn1 colonized host tissue as effectively as wild type strain 388, but histological examination showed that strain 388 exs1::Tn1 did not elicit as severe lung tissue pathology as that caused by wild type strain 388. See Nicas, et al., Eur. J. Clin. Microbiol. 4(2):175-9 (1985). Woods and colleagues, Woods, et al., Euro. J. Clin. Microbiol. 4(2):163-9 (1985), observed similar results in the rat lung model, utilizing transposon mutants of Pseudomonas aeruginosa strain DG1 that possessed a negative exoenzyme S phenotype. Together, these data have implicated exoenzyme S as a mediator of Pseudomonas aeruginosa dissemination from localized infections and have targeted exoenzyme S as a potential protective immunogen for vaccine development against Pseudomonas aeruginosa infections.
Exoenzyme S appears to be secreted into the culture medium as both a 53 kDa form and a 49 kDa form. See Nicas, et al., Infect. Immun. 45(2):470-4 (1984). The 49 kDa form of exoenzyme S possesses enzymatic activity following elution from sodium dodecyl sulfate (SDS)-polyacrylamide gels (PAGs), Coburn, et al., J. Biol. Chem. 266(10):6438-46 (1991) and Nicas, et al., Infect. Immun. 45(2):470-4 (1984), and has been designated the enzymatically active form of exoenzyme S. The 53 kDa form of exoenzyme S, which does not possess any apparent ADP-ribosyltransferase activity in vitro, Nicas, et al., Infect. Immun. 45(2):470-4 (1984), appears to be related to the 49 kDa form of exoenzyme S based upon apparent immunological cross-reactivity, Nicas, et al., Infect. Immun. 45(2):470-4 (1984) and Kulich, et al., Infect. Immun. 61(1):307-13 (1993); possession of a common amino-terminal amino acid sequence, Coburn, In Aktories (ed.), Current topics in microbiology and immunology:ADP-ribosylating toxins. 175:133-143 (1992) Springer-Verlag, Berlin; and sharing of common peptides following proteolytic and cyanogen bromide cleavage, Iglewski, 249-265 In Hardegree M. C. and Tu, A. T. (eds), Handbook of toxins, vol. 4, Marcel-Dekker, New York (1988). The absolute biochemical and genetic relationship between the 53 kDa form and the 49 kDa form of exoenzyme S has not been resolved. These data are consistent with the 53 kDa form and the 49 kDa form of exoenzyme S existing in a precursor-proteolytic product relationship and that the 53 kDa form of exoenzyme S undergoes a carboxyl-terminal cleavage to yield the enzymatically active 49 kDa form of exoenzyme S. The observation that other bacterial ADP-ribosyltransferases undergo proteolytic activation lends some precedence to this hypothesis. See Drazin, et al., J. Biol. Chem. 246(5):1504-10 (1971); Mekalanos, et al., J. Biol. Chem. 254(13):5855-61 (1979); and Vasil, et al., Infect. I Immun. 16(1):353-61 (1977).
The present invention focuses on the biochemical and molecular characterization of exoenzyme S as an ADP-ribosyltransferase. As prior exoenzyme S studies have relied on denatured, mixed preparations of the 49 and 53 kDa form, we are the first to show conclusively that exoenzyme S activity copurifies with two proteins possessing apparent molecular masses of 53 and 49 kDa respectively and that .alpha.-49 kDa protein IgG inhibits exoenzyme S mediated ADP-ribosyltransferase activity in a dose-dependent fashion Kulich, et al., Infect. Immun. 61(1):307-13 (1993). (This article is incorporated by reference as if fully set forth herein.) In the present invention, we also disclose the purification and proteolytic characterization of the 49 kDa form of exoenzyme S and the cloning of the structural gene for the 49 kDa form of exoenzyme S (exoS).